System and method for detecting of alpha-methylacyl-CoA racemase (AMACR) and prostate cancer

ABSTRACT

A detection system for determining alpha-methylacyl-CoA (AMACR) levels in a bodily sample includes at least one reaction solution for generating H 2 O 2  upon combination with AMACR in the bodily sample and a biosensor for determining a level of generated H 2 O 2 . The reaction solution includes a (2R)-2-methylacyl-CoA epimer that can be chirally inverted by AMACR to a (2S)-2-methylacyl-CoA epimer and an enzyme that carries out beta oxidation with the (2S)-2-methylacyl-CoA epimer to generate hydrogen peroxide (H 2 O 2 ).

RELATED APPLICATION

The present application is a U.S. National Stage under 35 USC 371 patentapplication, claiming priority to Serial No. PCT/US2013/056635, having afiling date of Aug. 26, 2013, which claims priority from U.S.Provisional Application No. 61/692,988, filed Aug. 24, 2012, the subjectmatter of which is incorporated herein by reference in its entirety.

BACKGROUND

Prostate cancer is the most common malignancy among men in the UnitedStates and also ranks as the second most common cause of cancer death inmales. The prostate specific antigen (PSA) blood test, in addition tothe digital rectal exam, have traditionally been the preferredmodalities to screen for prostate cancer. While prostate cancerscreening leads to an early diagnosis of prostate cancer, which in turnpermits curative treatment, it also has significant limitations becauseserum PSA is not specific to prostate cancer. One of the majorlimitations of PSA screening is that serum PSA can be elevated inpatients with other common benign conditions, such as benign prostatichyperplasia, prostatitis, or after minor clinical procedures, such astransrectal ultrasound. Accordingly, for every four men that haveprostate biopsies, one case of prostate cancer is detected.

PSA is also not a reliable biomarker for aggressive prostate cancer.High grade prostate cancers may actually produce less PSA and theabsolute PSA number does not accurately reflect the aggressiveness ofdisease. For men diagnosed with prostate cancer many have clinicallyinsignificant disease, which will never become symptomatic in theirlifetime. This “over-diagnosis” of clinically insignificant prostatecancer from PSA screening has been estimated to be as high as 30% withsubsequent over-treatment. The side effects of prostate cancer treatmentmay include unnecessary painful biopsies, surgical complications,radiation burns, incontinence, erectile dysfunction, bowel injury, andpatient anxiety. Thus, there is a clear need for an improved biomarkerfor prostate cancer.

Alpha-methylacyl-CoA racemase (AMACR) is an enzyme involved inperoxisomal beta-oxidation of dietary branched-chained fatty acids.AMACR has been consistently overexpressed in prostate cancer epithelium;hence it becomes an ideal specific biomarker for cancer cells within theprostate gland. Over-expression of AMACR may increase the risk ofprostate cancer, because its expression is increased in premalignantlesions (prostatic intraepithelial neoplasia). Furthermore,epidemiologic, genetic and laboratory studies have pointed to theimportance of AMACR in prostate cancer. Genome-wide scans of linkage inhereditary prostate cancer families have demonstrated that thechromosomal region for AMACR (5p 13) is the location of a prostatecancer susceptibility gene and AMACR gene sequence variants(polymorphisms) have been shown to co-segregate with cancer of theprostate in families with hereditary prostate cancer.

SUMMARY

Embodiments described herein relate to a detection system and in vitroassay for detecting, identifying, quantifying, and/or determining thelevel of alpha-methylacyl-CoA racemase (AMACR) in a bodily sample aswell as to a detection system and in vitro assay for diagnosing,identifying, staging, and/or monitoring prostate cancer in a subjecthaving, suspected of having, or at risk of prostate cancer. Thedetection system includes at least one reaction solution for generatingH₂O₂ upon combination with AMACR in the bodily sample and a biosensorfor determining the level of the generated H₂O₂. In some embodiments,the at least one reaction solution includes a (2R)-2-methylacyl-CoAepimer that can be chirally inverted by AMACR to a (2S)-2-methylacyl-CoAepimer and an enzyme that carries out beta oxidation with the(2S)-2-methylacyl-CoA epimer to generate hydrogen peroxide (H₂O₂). Inother embodiments, the reaction solution can include coenzyme A (CoA),peroxisomalacyl-coenzyme A oxidase 3 (ACOX3), adensonsine triphosphate(ATP), and a branched fatty acid with (R) and (S) epimers of which onlythe (R) epimer is a reaction substrate for AMACR. The branched fattyacid can be, for example, a 2-methyl carboxylic acid, which includes a(2R)-2-methyl carboxylic acid epimer and a (2S)-2-methyl carboxylic acidepimer. In one example, the branched fatty acid can be pristanic acid.

In some embodiments, the bodily sample can include a bodily fluid, suchas blood, plasma, sera, or urine, which can potentially include AMACR.

In other embodiments, the biosensor can include a working electrode, acounter electrode, and optionally a reference electrode. The workingelectrode and counter electrode can include catalyst particles that canincrease the rate of electrochemical oxidation-reduction reaction withH₂O₂ and provide for the detection of H₂O₂ at a lower oxidationpotential than without the presence of the catalyst particles. Thecatalyst particles can include nano-particle metallic catalysts, such asa unary metal (M), a binary metal (M-X), a unary metal oxide (MOy), abinary metal oxide (MOy-XOy), a metal-metal oxide composite material(M-MOy) or a combination of which, wherein y is less than 3, and M and Xare independently selected from a group consisting of Li, Na, Mg, Al, K,Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Sr, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag,Cd, In, Sn, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Lu, Ta,W, Os, Ir, Pt, Au, and Pb. In one embodiment, the catalyst particles cancomprise iridium oxide particles.

The detection system can also include a measuring device for applying avoltage potential to the working electrode, counter electrode, and/orreference electrode and measuring the current flow between the workingelectrode and counter electrode.

Other embodiments described herein relate to a method of detecting,identifying, quantifying, and/or determining the level of AMACR in abodily sample as well as a method for diagnosing, identifying, staging,and/or monitoring prostate cancer in a subject having, suspected ofhaving, or at risk of prostate cancer. The method includes obtaining abodily sample from the subject. The bodily sample can include a bodilyfluid, such as blood, plasma, sera, and urine, which can potentiallyinclude AMACR. The bodily sample is combined with at least one reactionsolution for generating H₂O₂ upon combination with AMACR in the bodilysample. In some embodiments, the at least one reaction solution includesa (2R)-2-methylacyl-CoA epimer that can be chirally inverted by AMACR to(2S)-2-methylacyl-CoA epimer and an enzyme that carries out betaoxidation with the (2S)-2-methylacyl-CoA epimer to generate hydrogenperoxide (H₂O₂). In other embodiments, the reaction solution can includecoenzyme A (CoA), peroxisomalacyl-coenzyme A oxidase 3 (ACOX3),adensonsine triphosphate (ATP), and a branched fatty acid with (R) and(S) epimers of which only the (R) epimer is a reaction substrate forAMACR. The branched fatty acid can be, for example, a 2-methylcarboxylic acid, which includes a (2R)-2-methyl carboxylic acid epimerand a (2S)-2-methyl carboxylic acid epimer. In one example, the branchedfatty acid can be pristanic acid. The quantity, amount, or level of H₂O₂generated in the reaction solution is detected with a biosensor. Anincreased amount of H₂O₂ detected compared to a control is indicative ofan increased amount level of AMACR in the bodily sample and indicativeof the subject having prostate cancer or an increase risk of prostatecancer.

In some embodiments, the biosensor can include a working electrode and acounter electrode, and optionally a reference electrode. The workingelectrode and counter electrode can include catalyst particles that canincrease the rate of electrochemical oxidation-reduction reaction withH₂O₂ and provide for the detection of H₂O₂ at a lower oxidationpotential than without the presence of the catalyst particles. Thecatalyst particles can include nano-particle metallic catalysts, such asa unary metal (M), a binary metal (M-X), a unary metal oxide (MOy), abinary metal oxide (MOy-XOy), a metal-metal oxide composite material(M-MOy) or a combination of which, wherein y is less than 3, and M and Xare independently selected from a group consisting of Li, Na, Mg, Al, K,Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Sr, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag,Cd, In, Sn, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Lu, Ta,W, Os, Ir, Pt, Au, and Pb. In one embodiment, the catalyst particles cancomprise iridium oxide particles.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of a biosensor in accordance with anaspect of the application.

FIG. 2 is a top plan view of an array of biosensors in a rowmanufactured by a screen-printing process.

FIGS. 3(A-C) illustrates plots showing cyclic voltammograms of (a)control sample solutions of PBS media and PB+pristanic acid; (b) PBS andPBS+AMACR; and (c) pristanic acid+PBS+ACOX3+AMACR, pristanicacid+PBS+ACOX3, and pristanic acid and PBS.

FIGS. 4(A-B) illustrate plots showing measured current readings fromdifferent AMACR containing plasma samples in (A) chronological order and(B) sample numbering order. Samples 1-9 are from healthy men. Samples11-20 are from men with high grade prostatic intraepithelial neoplasia(HGPIN). Samples 21-25 are from men with prostate cancer (Gleason score6-7).

DETAILED DESCRIPTION

Unless specifically addressed herein, all terms used have the samemeaning as would be understood by those of skilled in the art of thesubject matter of the application. The following definitions willprovide clarity with respect to the terms used in the specification andclaims.

As used herein, the term “monitoring” refers to the use of resultsgenerated from datasets to provide useful information about anindividual or an individual's health or disease status. “Monitoring” caninclude, for example, determination of prognosis, risk-stratification,selection of drug therapy, assessment of ongoing drug therapy,determination of effectiveness of treatment, prediction of outcomes,determination of response to therapy, diagnosis of a disease or diseasecomplication, following of progression of a disease or providing anyinformation relating to a patient's health status over time, selectingpatients most likely to benefit from experimental therapies with knownmolecular mechanisms of action, selecting patients most likely tobenefit from approved drugs with known molecular mechanisms where thatmechanism may be important in a small subset of a disease for which themedication may not have a label, screening a patient population to helpdecide on a more invasive/expensive test, for example, a cascade oftests from a non-invasive blood test to a more invasive option such asbiopsy, or testing to assess side effects of drugs used to treat anotherindication.

As used herein, the term “quantitative data” or “quantitative level” or“quantitative amount” refers to data, levels, or amounts associated withany dataset components (e.g., markers, clinical indicia,) that can beassigned a numerical value.

As used herein, the term “subject” refers to a male human or anothermale mammal, which can be afflicted by a prostate disease, includingprostate cancer, but may or may not have such a disease. Typically, theterms “subject” and “patient” are used herein interchangeably inreference to a human individual.

As used herein, the term “subject suspected of having prostate cancer”refers to a subject that presents one or more symptoms indicative ofprostate cancer or that is being screened for prostate cancer (e.g.,during a routine physical examination). A subject suspected of havingprostate cancer may also have one or more risk factors. The termencompasses individuals who have not been tested for prostate cancer,individuals who have received an initial diagnosis (e.g., a CT scanshowing a mass) but for whom the stage of cancer is not known, as wellas individuals for whom the stage and/or grade of cancer has beendetermined by a conventional method (e.g., Gleason score). The term alsoincludes patients who have previously undergone therapy for prostatecancer, including radical prostatectomy and brachytherapy.

As used herein, the term “subject at risk for prostate cancer” refers toa subject with one or more risk factors for developing prostate cancer.Risk factors include, but are not limited to, gender, age, geneticpredisposition, previous incidents with cancer, and pre-existingnon-cancer diseases.

As used herein, the term “diagnosing prostate cancer” or “detectingprostate cancer” refers to a process aimed at one or more of:determining if a subject is afflicted with prostate cancer; determiningthe severity or stage of prostate cancer in a subject; determining therisk that a subject is afflicted with prostate cancer; and determiningthe prognosis of a subject afflicted with prostate cancer.

As used herein, the term “subject diagnosed with prostate disease”refers to a subject who has been tested and found to have prostatedisease. The diagnosis may be performed using any suitable method,including, but not limited to, biopsy, x-ray, blood test, and themethods described herein.

As used herein, the term “providing a prognosis” refers to providinginformation regarding the impact of the presence of prostate cancer(e.g., as determined by the methods described herein) on a subject'sfuture health. Providing a prognosis may include predicting one or moreof: prostate cancer progression, the likelihood of prostatecancer-attributable death, the average life expectancy of the patient,the likelihood that the patient will survive for a given amount of time(e.g., 6 months, 1 year, 5 years, etc), the likelihood that the patientwill be disease-free for a specified prolonged period of time, thelikelihood of getting prostate cancer, the likelihood of developingaggressive prostate cancer, the likelihood of recurrence, and the riskof metastasis. In certain embodiments, the prognosis methods describedherein are used clinically to make treatment decisions by choosing themost appropriate treatment modalities for any particular patient.

As used herein, the term “bodily sample” refers to a sample that may beobtained from a subject (e.g., a human) or from components (e.g.,tissues) of a subject. The sample may be of any biological tissue orfluid with which biomarkers described herein may be assayed. Frequently,the sample will be a “clinical sample”, i.e., a sample derived from apatient. Such samples include, but are not limited to, bodily fluids,e.g., urine, blood, plasma, or sera; and archival samples with knowndiagnosis, treatment and/or outcome history. The term biological samplealso encompasses any material derived by processing the biologicalsample. Processing of the bodily sample may involve one or more of,filtration, distillation, extraction, concentration, inactivation ofinterfering components, addition of reagents, and the like.

As used herein, the terms “normal” and “healthy” are usedinterchangeably. They refer to an individual or group of individuals whohave not shown any symptoms of prostate cancer, and have not beendiagnosed with prostate cancer. Preferably, the normal individual (orgroup of individuals) is not on medication affecting prostate cancer. Incertain embodiments, normal individuals have similar sex, age, body massindex as compared with the individual from which the sample to be testedwas obtained. The term “normal” is also used herein to qualify a sampleisolated from a healthy individual.

As used herein, the terms “control” or “control sample” refer to one ormore biological samples isolated from an individual or group ofindividuals that are normal (i.e., healthy). The term “control”,“control value” or “control sample” can also refer to the compilation ofdata derived from samples of one or more individuals classified asnormal, and/or one or more individuals diagnosed with prostate cancer.

As used herein, the term “indicative of prostate cancer”, when appliedto an amount of at least one alpha-methylacyl-CoA racemase (AMACR) in abodily sample, refers to a level or an amount, which is diagnostic ofprostate cancer such that the level or amount is found significantlymore often in subjects with the disease than in subjects without thedisease or another stage of prostate cancer (as determined using routinestatistical methods setting confidence levels at a minimum of 95%).Preferably, a level or amount, which is indicative of prostate cancer,is found in at least about 60% of subjects who have the disease and isfound in less than about 10% of subjects who do not have the disease.More preferably, a level or amount, which is indicative of prostatecancer, is found in at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95% ormore in subjects who have the disease and is found in less than about10%, less than about 8%, less than about 5%, less than about 2.5%, orless than about 1% of subjects who do not have the disease.

Embodiments described herein relate to detection systems, in vitroassays, and/or methods for detecting, identifying, quantifying, and/ordetermining the level of alpha-methylacyl-CoA racemase (AMACR) in abodily sample and to detection systems, in vitro assays, and/or methodsfor diagnosing, identifying, staging, and/or monitoring prostate cancerin a subject having, suspected of having, or at risk of prostate cancer.

The detection systems and methods described herein provide a single use,disposable, and cost-effective biosensor for simple point-of-care andearly detection of prostate cancer using bodily samples, such as bodilyfluids obtained by non-invasive or minimally invasive means, whichminimizes complicated clinical procedures for cancer screening. Currentassays for the detection of AMACR, a protein that has been identified tocorrelate with the occurrence of prostate cancer, have not beeneffective due to the limited understanding of its chemical detection. Inthis application, a detection and in vitro assay is provided that canuse electrocatalysts to enhance the sensitivity of an electrochemicalbiosensor that can screen bodily samples, such as bodily fluids,including blood, sera, plasma or urine samples, for the detection,diagnosis, identification, staging, and/or monitoring of prostatecancer.

The detection systems and assays or methods described herein include atleast one reaction solution that can be used to generate a detectableand/or quantifiable analyte, which is indicative of the amount,concentration, or level of AMACR in a bodily sample of a subjectsuspected of having or at risk of prostate cancer, and a biosensor fordetecting the amount, level, or concentration of the analyte in thereaction solution. The components of the reaction solution are based ona biochemical pathway that necessitates the participation of AMACR inthe mechanistic sequence. The reaction solution includes a(2R)-2-methylacyl-CoA epimer that can be chirally inverted by AMACR inthe bodily fluid to (2S)-2-methylacyl-CoA epimer and an enzyme thatcarries out beta oxidation with the formed (2S)-2-methylacyl-CoA epimerto generate an analyte, hydrogen peroxide (H₂O₂). The amount,concentration, or level of H₂O₂ generated by biochemical reaction of thereaction sample and AMACR in the bodily sample obtained from the subjectsuspected of having or at risk of prostate cancer can be measured usingthe biosensor to determine the amount, concentration, or level of AMACRin the bodily fluid and hence whether the subject has prostate cancer oran increased risk of prostate cancer.

In some embodiments, the at least one reaction solution includes abranched fatty acid with (R) and (S) epimers of which only the (R)epimer can form a reaction substrate for AMACR. The branched fatty acidcan be, for example, a branched fatty acid, which includes a(2R)-2-methyl carboxylic acid epimer and a (2S)-2-methyl carboxylic acidepimer. In one example, the branched fatty acid can be pristanic acid.Pristanic acid possesses four characteristic carbon atoms in itschemical backbone. The carbon atom in the second position, C-2, willyield either (R)-configuration or (S-)configuration epimers indicated as(2R) and (2S) in the reaction scheme below.

As shown in the reaction scheme above, both (2R) and (2S) epimers canreact with a proper quantity of co-enzyme A (CoA), ATP and Mg⁺², whichcan also be provided in the reaction solution, forming(2R)-pristanoyl-CoA and (2S)-pristanoyl-CoA, respectively.(2R)-pristanoyl-CoA cannot carry out a beta oxidation process to produceH₂O₂. Conversely, as shown below, (2S)-pristanoyl-CoA in the presence ofthe enzyme ACOX₃ (peroxisomalacyl-coenzyme A oxidase 3) can carry out abeta-oxidation process producing H₂O₂, which can be detectedelectrochemically.

Therefore, a sensor, which can measure the generated H₂O₂,quantitatively, can be used to measure the quantity of the(2S)-pristanoyl-CoA.

(2R)-pristanoyl-CoA in the presence of AMACR, as shown below, willconvert to (2S)-pristanoyl-CoA that can then be oxidized to produce moreH₂O₂ in the presence of peroxisomalacyl-coenzyme A oxidase 3 (ACOX3).

The additional H₂O₂ produced due to the AMACR conversion of(2R)-pristanoyl-CoA to (2S)-pristanoyl-CoA can be quantified andcompared to a control value or level to determine the quantity of AMACRin a bodily sample. The quantity of AMACR in the bodily sample obtainedfrom a subject suspected of having or at risk of prostate cancer candirectly affect the production of (2S)-pristanoyl-CoA and hence H₂O₂production. Thus, the quantified level of H₂O₂ generated can be comparedto a control or predetermined value to determine, the level of AMACR inthe bodily sample, and if or whether the subject has prostate cancer.For example, an increase in the detected level of H₂O₂ in a bodilysample mixed with the reaction solution compared to a control value isindicative of the subject having prostate cancer or an increased risk ofprostate cancer.

In some embodiments, the at least one reaction solution with which thebodily sample obtained from the subject is mixed can include a pristanicacid solution, adensonsine triphosphate (ATP), magnesium chloride,coenzyme A (CoA), and peroxisomalacyl-coenzyme A oxidase 3 (ACOX3). Byway of example, in preparing the reaction solution, a pristanic acidsolution, purchased from Sigma-Aldrich, can be mixed with phosphatebuffered saline solution (PBS), so that the volume ratio between the PBSand pristanic acid solution is about 1:1. PBS solution with a pH of 6.5can be prepared by mixing monobasic and dibasic sodium phosphates withdeionized water, and 200 mM of potassium chloride can be added as thesupporting electrolyte to improve conductivity of the buffer. 3 mg ofATP, 3 mg of CoA, and 3 mg of magnesium chloride can also be added tothe PBS/pristanic acid mixture. 5 μl of this solution can then combinedwith 1 μl of ACOX3 to form the reaction solution. Advantageously, thereaction solution does not include any reagents or byproducts that wouldpotentially contribute to background oxidation current of the biosensorand impair detection and quantification of the H₂O₂ generated.

The reaction solution so formed can be mixed with a bodily sample, suchas a bodily fluid (e.g., blood, sera, plasma, or urine), obtained fromthe subject. In some aspects, the amount of blood taken from a subjectis about 0.1 ml or more and the amount added to about 6 μl of thereaction solution can be about 1 μl or less. In some embodiments, thebodily sample is blood plasma isolated from a whole blood sampleobtained from a subject. Blood plasma may be isolated from whole bloodusing well known methods, such as centrifugation.

The bodily samples can be obtained from the subject using samplingdevices, such as syringes, swabs or other sampling devices, used toobtain liquid and/or solid bodily samples either invasively (i.e.,directly from the subject) or non-invasively. These samples can then bestored in storage containers. The storage containers used to contain thecollected sample can include a non-surface reactive material, such aspolypropylene. The storage containers are generally not made fromuntreated glass or other sample reactive material to prevent the samplefrom becoming absorbed or adsorbed by surfaces of the glass container.

Collected samples stored in the container may be stored underrefrigeration temperature. For longer storage times, the collectedsample can be frozen to retard decomposition and facilitate storage. Forexample, samples obtained from the subject can be stored in a falcontube and cooled to a temperature of about −80°.

The H₂O₂, which is generated by mixing of the bodily sample containingAMACR with the reaction solution, is an electrochemically active speciesthat can be oxidized or reduced under appropriate conditions anddetected using an H₂O₂ biosensor to quantify the level of AMACR in thebiological sample and determine whether the subject has or is at risk ofprostate cancer. In some embodiments, the H₂O₂ biosensor can include atwo or three electrode electrochemical biosensor. The biosensor can bemanufactured by established micro-fabrication techniques, includingthick film screen printing, ink jet printing, or laser etchingprocesses. This fabrication process can also use a combination of theseand any other fabrication techniques. Advantageously, the H₂O₂ biosensorcan be produced using thick film screen print. Thick film screenprinting provides a cost-effective, single use, disposable biosensorminimizing any electrode cleaning, sterilization and electrode foulingproblems.

FIG. 1 illustrates an H₂O₂ biosensor 10 in accordance with an embodimentof the application. The biosensor 10 is a three-electrode sensorincluding a counter electrode 12, a working electrode 14, and areference electrode 16, which are exposed to the reaction solution in adetection region 20 of the biosensor 10. A voltage source 22 isconnected to the working and reference electrodes 14, 16. A currentmeasuring device 24 is connected to the working and counter electrodes14, 12 to measure the current generated by the redox reaction of H₂O₂when the mixture of reaction solution and biological sample is added tothe detection region 20 of the biosensor 10.

The working electrode 14 is the site of the redox reaction of H₂O₂, andwhere the charge transfer occurs. The function of the counter electrode12 is to complete the circuit, allowing charge to flow through thesensor 10. The working electrode 14 and the counter electrode 12 arepreferably formed of the same material, although this is not arequirement. Examples of materials that can be used for the workingelectrode 14 and counter electrode 12 include, but are not limited to,gold, platinum, palladium, silver, and carbon.

Examples of materials that can be used to form the reference electrode16 are silver-silver chloride and mercury-mercuric chloride (Calomel).Silver-silver chloride is preferred. The silver can be applied to asubstrate in the form of a silver ink, which is commercially available,or can be made using finely dispersed metal particles, solvent, and abinder. Respective silver contact pads 30, 32, and 34 are connected witheach of the electrodes 12, 14, and 16. An insulation layer 40 may coverpart of the electrodes 12, 14, and 16, leaving tips of the electrodes12, 14, and 16 exposed to the detection environment in the detectionregion 20.

In some embodiments, the working and counter electrodes 14, 12 caninclude a layer of particles, such as micro-, meso- or nano-sizedparticles of active carbon or porous carbon. The active carbonnanoparticles may be combined with metallic catalyst particles thatincrease the rate of electrochemical oxidation-reduction reaction withH₂O₂ and provide the detection of H₂O₂ at a lower oxidation potentialthan without the presence of the catalyst particles. In terms of thepractical applications, the metallic catalyst particles can shorten thereaction time and lower the applied electrochemical potential fordetection of H₂O₂ in the mixture of the reaction solution and biologicalsample. Lowering the applied potential often leads to the minimizationof electrochemical oxidation or reduction of other species presented,resulting in a minimization of interference caused by the unwantedreaction of the confounding species. As a result, a highly specificbiosensor can be obtained and produced.

The metallic catalyst particles can include nano-, meso-, or micro-scaleparticles of a unary metal (M), a binary metal (M-X), a unary metaloxide (MOy), a binary metal oxide (MOy-XOy), a metal-metal oxidecomposite material (M-MOy) or a combination of which, wherein y is lessthan 3, and M and X are independently selected from a group consistingof Li, Na, Mg, Al, K, Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Sr, Y, Zr, Nb,Mo, Ru, Rh, Pd, Ag, Cd, In, Sn, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy,Ho, Er, Tm, Lu, Ta, W, Os, Ir, Pt, Au, and Pb. In one embodiment, forexample, the metallic catalyst particles may be composed of a unarymetal, unary metal oxide binary metal, or binary metal oxide, such asiridium, iridium oxide, platinum, ruthenium, platinum-ruthenium,platinum-nickel, and platinum-gold.

In one example, the working electrode 14 and the counter electrode 12can be made of active carbon and include about 2 to about 5 weightpercent iridium oxide nanoparticles. Incorporation of about 2 to about 5weight percent iridium oxide nanoparticles into the working electrodeand counter electrode can lower oxidation potential of H₂O₂ in themixture of reaction solution and biological to 0.25 Volt versus astandard Ag/AgCl reference electrode from about 0.40 to about 0.45 Volt.At this lower potential, oxidation of other biological species in themixture of the reaction solution and biological can be minimized.

The biosensor illustrated in FIGS. 1 and 2 can be fabricated on asubstrate 100 formed from polyester or other electrically non-conductivematerial, such as other polymeric materials, alumina (Al₂O₃), ceramicbased materials, glass or a semi-conductive substrate, such as silicon,silicon oxide and other covered substrates. Multiple sensor devices 102can thus be formed on a common substrate 100 (FIG. 2). As will beappreciated, variations in the geometry and size of the electrodes arecontemplated.

The biosensor can be made using a thin film, thick film, and/or ink-jetprinting technique, especially for the deposition of multiple electrodeson a substrate. The thin film process can include physical or chemicalvapor deposition. Electrochemical sensors and thick film techniques fortheir fabrication are discussed in U.S. Pat. No. 4,571,292 to C. C. Liuet al., U.S. Pat. No. 4,655,880 to C. C. Liu, and co-pending applicationU.S. Ser. No. 09/466,865, which are incorporated by reference in theirentirety. By way of example, in the case of the carbon electrodes,active carbon is mixed with a binder, deposited like an ink on thesubstrate, and allowed to dry.

The voltage source can apply a voltage potential to the workingelectrode 14 and reference and/or counter electrode 16, 12, depending onthe design of the biosensor 10. The current between the workingelectrode 14 and counter electrode 16 can be measured with a measuringdevice or meter. Such current is due to the reduction occurring at theworking electrode 12 of H₂O₂ generated by AMACR in the bodily samplethat is mixed and/or combined with the reaction solution.

The amount or level of current measured is proportional to the amount ofH₂O₂ generated and the level or amount of AMACR in the bodily sample aswell as the risk or presence of prostate cancer in the subject. Once thecurrent level generated by the bodily sample tested with the biosensoris determined, the level can be compared to a predetermined value orcontrol value to provide information for diagnosing or monitoring ofprostate cancer in a subject. For example, the current level can becompared to a predetermined value or control value to determine if asubject is afflicted with or has prostate cancer. An increased currentlevel compared to a predetermined value or control value can beindicative of the subject having prostate cancer; whereas similar ordecreased current level compared to a predetermined value or controlvalue can be indicative of the absence of prostate cancer in the subject

The current level generated by the bodily sample obtained from thesubject can be compared to a current level of a bodily sample previouslyobtained from the subject, such as prior to administration of atherapeutic. Accordingly, the methods described herein can be used tomeasure the efficacy of a therapeutic regimen for the treatment ofprostate cancer in a subject by comparing the current level obtainedbefore and after a therapeutic regimen. Additionally, the methodsdescribed herein can be used to measure the progression of prostatecancer in a subject by comparing the current level in a bodily sampleobtained over a given time period, such as days, weeks, months, oryears.

The current level generated by a bodily fluid of the subject may also becompared to a predetermined value or control value to provideinformation for determining the severity or aggressiveness of theprostate cancer in the subject. Thus, in some aspect, the current levelmay be compared to control values obtained from subjects with well knownclinical categorizations, or stages of histopathologies related toprostate cancer (e.g., Gleason score of prostate cancer or indolentversus aggressive prostate cancer). In one particular embodiment, thecurrent in a sample can provide information for determining a particularGleason grade or score of prostate cancer in the subject.

A predetermined value or control value can be based upon the currentlevel in comparable samples obtained from a healthy or normal subject orthe general population or from a select population of control subjects.In some aspects, the select population of control subjects can includeindividuals diagnosed with prostate cancer. For example, a subjecthaving a greater current level compared to a control value may beindicative of the subject having a more advanced stage of a prostatecancer.

The select population of control subjects may also include subjectsafflicted with prostate cancer in order to distinguish subjectsafflicted with prostate cancer from those with benign prostate disease.In some aspects, the select population of control subjects may include agroup of individuals afflicted with prostate cancer.

The predetermined value can take a variety of forms. The predeterminedvalue can be a single cut-off value, such as a median or mean. Thepredetermined value can be established based upon comparative groupssuch as where the current level in one defined group is double thecurrent level in another defined group. The predetermined value can be arange, for example, where the general subject population is dividedequally (or unequally) into groups, or into quadrants, the lowestquadrant being subjects with the lowest current level, the highestquadrant being individuals with the highest current level. In anexemplary embodiment, two cutoff values are selected to minimize therate of false positive and negative results.

The Example that follows illustrates embodiments of the presentinvention and are not limiting of the specification and claims in anyway.

Example 1

In this Example we present the results of the development of a biosensorfor the detection of AMACR in human serum samples. We tested thebiosensor for the ability to identify patients with prostate cancer. Wefound, using this biosensor with plasma samples from 24 men, that wewere able to distinguish, with 100% accuracy, between both healthy men,men with high grade prostatic intraepithelial neoplasia, and men withbiopsy proven prostate cancer.

Methods

Chemistry and Reaction Mechanisms of Detecting AMACR

Pristanic acid possesses four methyl groups. Based on the reactionscheme above, pristanic acid can be employed as reaction substrate whichconsists of two epimers designated as (2R) and (2S). Both (2R) and (2S)epimers can react with proper quantities of coenzyme A (CoA), ATP andMg²⁺ in the presence of very long chain fatty acid-coenzyme A(VLCFA-CoA) synthetase forming (2R)-pristanoyl-CoA and(2S)-pristanoyl-CoA, respectively. However, the (2R)-pristanoyl-CoAcannot carry out the β-oxidation process. On the other hand,(2S)-pristanoyl-CoA in the presence of the enzyme ACOX3(peroxisomalacyl-coenzyme A oxidase 3) can carry out the β-oxidationprocess producing H₂O₂. H₂O₂ can be oxidized electrochemicallygenerating a current which can then be quantified (2S)-pristanoyl-CoAAMACR converts (2R)-pristanoyl-CoA to (2S)-pristanoyl-CoA, resulting ina higher H₂O₂ level, and the oxidation current of H₂O₂ can then be usedto quantify the amount of AMACR present.

AMACR Biosensor Fabrication

This biosensor was thick film printed on 0.18 mm thick polyethyleneterephalate (PET) substrate (Melinex 329, DuPont Co.) in the dimensionof 385×280 mm². The cost of this thick film process was relatively low,and 150 individual biosensors were produced in 6 rows per sheet. Thebiosensor had a three-electrode configuration: working, counter, andreference electrode. Both the working and counter electrodes wereprinted with nanoparticles of iridium (actually IrO) contained activecarbon power (RC72), which was mixing into a thick-film printing ink.The reference electrode was also a thick film printed Ag/AgCl electrodeusing DuPont #5870 Ag/AgCl thick film ink. The insulation layer wasthick-film printed using Nazdar APL 34 silicone-free dielectric ink,which also defined the dimensions of the individual biosensor. Theworking electrode was approximately 0.8 mm² with a diameter of 1.0 mm.The biosensor could accommodate 10-20 micro-liter of test volume. Otherelectrocatalysts have been tested and evaluated in addition to IrOnano-catalyst.

It must be recognized that this biosensor prototype was fabricated bythick-film printing technology, the manufacturing cost can be relativelylow making it to be a single use, disposable biosensor a reality.However, thick film printing technology has an inherent accuracylimitation of 10%. Therefore, this limitation must be included in theconsideration of the biosensor measurement.

FIGS. 1 and 2 illustrate schematically a biosensor prototype, which wasdeveloped in our laboratory. This biosensor can be manufactured costeffectively using thick film screen printing techniques. This biosensorincorporates a nanoparticle catalyst, iridium oxide, lowering theoxidation electrochemical potential of H₂O₂ and minimizing oxidation ofother chemicals in the actual physiological fluid samples. This providesadditional sensitivity and selectivity to the biosensor. Using thisunique H₂O₂ biosensor and the AMACR assay approach shown in the reactionscheme above, we are able to establish an in vitro assay of AMACR inserum samples for effective prostate cancer detection and diagnosis.

Calibration of this AMACR Biosensor

Hydrogen peroxide, H₂O₂, is an electrochemically active species whichcan be oxidized or reduced under appropriate conditions. Normally, atapproximately +0.40 to 0.45 Volt versus a Ag/AgCl reference electrode,H₂O₂ can be oxidized yielding an oxidation current which corresponds tothe quantity of H₂O₂ presented. With the incorporation of the IrOnano-catalyst, the overpotential of the oxidation of H₂O₂ becomes lowerand H₂O₂ in PBS can then be oxidized at 0.25 V versus the Ag/AgClreference. This represents the advantage and uniqueness of thisbiosensor sensor. FIG. 4 shows the calibration curve of the biosensorrelating the oxidation current of the biosensor to the concentration ofthe H₂O₂. With the chemicals involved, such as pristanic acid, PBS,Mg⁺², CoA, ATP, ACOX3 and AMACR, the oxidation potential applied to theH₂O₂ produced was +0.4 V versus the Ag/AgCl reference. This valueremained lower than the oxidation potential of H₂O₂ in the presence ofthese chemicals, +0.6 V versus the Ag/AgCl reference electrode. Thisassessment was experimentally verified by cyclic voltammetric studies.

This approach and the reaction scheme shown above can only be applicablefor AMACR detection, if all the chemical species involved in thereactions will not contribute to any oxidation current of the H₂O₂produced. This can be validated by carrying out the cyclic voltammetricstudies of the chemicals involved. FIG. 3 shows the cyclic voltammetricstudies indicating that the chemicals used will not contribute to theoxidation current of hydrogen peroxide produced in by the reactionscheme shown above.

FIG. 3A shows the background electrochemical signal from the AMACRsubstrate, pristanic acid, in phosphate buffer saline (PBS),demonstrating that the substrate by itself does not contribute to anybackground current. Similarly, the enzyme, AMACR, does not contribute tothe background current. FIG. 3B shows the absence of detectablebackground current due to AMACR (0.0065 μg/μl) in PBS media. From themetabolic β-oxidation pathway of pristanic acid illustrated in FIG. 1,(2S)-pristanoyl-CoA cannot be oxidized without the presence of ACOX3(i.e., electrochemically detectable H₂O₂ will not form without ACOX3).This is verified in FIG. 3C, where the experimental results of theoxidation currents detected by the biosensor in the presence ofpristanic acid, pristanic acid+ACOX3, and pristanic acid+ACOX3+AMACR areshown. This increase in detected oxidation current due to the additionof AMACR is specific and unique for pristanoyl-CoA and free ofinterferences from other molecules. In FIG. 3C, the higher currentproduced as a result of the conversion of (2R)-pristanoyl-CoA by AMACRto (2S)-pristanoyl-CoA is evident. These results illustrate that theelectrochemical detection of AMACR is quantitative and selective.

This reaction required an incubation time period to produce H₂O₂.Various incubation times were used and assessed. We have chosen 48 hoursas the incubation time, and this length of time may be furtheroptimized.

Testing in Human Plasma Samples

Plasma samples were obtained from 24 patients at University HospitalsCase Medical Center. These samples included nine healthy volunteers, 10men with histologically confirmed HGPIN and 5 men with newly diagnosedprostate cancer. 5 mL of blood was collected for each patient instandard heparinized tubes. Plasma was isolated by standard protocolsand frozen until future use. All samples were collected prior totreatment, where relevant.

To quantify AMACR, samples were first thawed and 5 μl aliquots weremade. Pristanic acid (#P6617 Sigma-Aldrich) was mixed with phosphatebuffer solution (PBS) with a volumetric ratio of 1:1. PBS solution had apH value of 6.5 prepared by mixing the appropriate quantity of monobasicand dibasic sodium phosphates with deionized water. 200 mM of potassiumchloride was added into the PBS as the supporting electrolyte improvingthe conductivity of the buffer solution. 3 mg of adenosine triphosphateATP (#A1852 Sigma-Aldrich) 3 mg magnesium chloride (#208337Sigma-Aldrich) and 3 mg coenzyme A (CoA)(#C3144 Sigma-Aldrich) wereadded into the a total of 140 μl of the pristanic acid-PBS mixedsolution. This solution was incubated in −20 C for 72 hours prior to usein any testing.

The applied potential for the current measurement was set at +0.4 Vversus the Ag/AgCl reference. 5 μl of the prepared pristanic acid-PBSsolution was first mixed with 1 μl of peroxisomalacyl-coenzyme A oxidase3 (ACOX3) (#H00008310-Q01 Sigma-Aldrich) and then mixed with 1 μl of thehuman serum sample. This mixture was incubated for one hour at roomtemperature. 5 μl of this mixture was then drawn and placed on thebiosensor surface and the oxidation current measured. All samples wererun in triplicate. Laboratory personnel were blinded to the diseasestatus of the samples.

The mean of the triplicate runs for each sample was calculated torepresent the estimated quantity of AMACR in that sample. Sensitivityand Specificity was calculated using a cutoff as determined by the data.

Results

To test the practical application of this AMACR biosensor, measurementof the level of AMACR in human biological specimens was carried out. Weexpected that the level of AMACR would increase in prostate cancerpatients. In order to evaluate this assessment, plasma samples from 9healthy males, 10 men with high grade prostatic neoplasia (HGPIN) and 5men with prostate cancers were used in a laboratory-blinded test of theAMACR levels in these samples. 5 mL of blood was collected from eachpatient in standard heparinized tubes. Plasma was isolated by standardprotocols and frozen until future use. All samples were collected priorto treatment, where relevant.

Table 1 shows the characteristics of these patients and the averageAMACR levels. To quantify AMACR, samples were first thawed and 5 μLaliquots were made. Pristanic acid (#P6617 Sigma-Aldrich) was mixed withphosphate buffer solution (PBS) with a volumetric ratio of 1:1. PBSsolution had a pH value of 6.5 prepared by mixing the appropriatequantity of monobasic and dibasic sodium phosphates with deionizedwater. 200 mM of potassium chloride was added into the PBS as thesupporting electrolyte improving the conductivity of the buffersolution. 3 mg of adenosine triphosphate ATP (#A1852 Sigma-Aldrich) 3 mgmagnesium chloride (#208337 Sigma-Aldrich) and 3 mg coenzyme A (CoA)(#C3144 Sigma-Aldrich) were added into the a total of 140 μL of thepristanic acid-PBS mixed solution. This solution was incubated in −20°C. for 72 h prior to use in any testing.

TABLE 1 Population Description and Mean AMACR Levels by Patient GroupHealthy Controls HGPIN Prostate Cancer (N = 9) (N = 10) Cases (N = 5)Gleason score (N (%) N/A N/A 4 (80%) 3 + 3 1 (20%) 3 + 4 Mean (SD)Plasma 2.31 18.86 15.81 PSA, ng/mL (1.67) (7.43) (11.43) Mean (SD)Plasma 0.005 0.0004 0.077 AMACR, μg/μl (0.001) (0.0005) (0.10)

The applied potential for the current measurement was set at +0.4 Vversus the Ag/AgCl reference. 5 μL of the prepared pristanic acid-PBSsolution was first mixed with 1 μL of peroxisomalacyl-coenzyme A oxidase3 (ACOX3) (#H00008310-Q01 Sigma-Aldrich) and then mixed with 1 μL of thehuman serum sample. This mixture was incubated for one hour at roomtemperature (−21° C.). 5 μL of this mixture was then drawn and placed onthe biosensor surface and the oxidation current measured. All sampleswere run in triplicate. Laboratory personnel were blinded to the diseasestatus of the samples.

The mean of the triplicate runs for each sample was calculated torepresent the estimated quantity of AMACR in that sample. Sensitivityand specificity were calculated using a cutoff as determined by thedata.

FIG. 4 shows the level of AMACR as measured by our biosensor for eachsample. As illustrated in FIGS. 4A and 4B, using a current level cutoffof anywhere between 0.08 and 0.90 would provide 100% sensitivity and100% specificity to separate the prostate cancer cases from the otherpatients. Thus, in our preliminary data, the accuracy of this test is100%.

Importantly, this detection method clearly distinguishes prostate cancerpatients not only from healthy men, but also from men with HGPIN, whichis a common limitation of other prostate cancer biomarkers. The currentoutput of this detection method is one order of magnitude higher forprostate cancer patients compared to that for either healthy or HGPINmales. This large difference in current outputs for the biosensorsprovides accuracy in distinguishing the prostate cancer patients fromthe normal and benign individuals. The findings thus far demonstrate thedetection of AMACR level using this relatively simple andminimally-invasive (not requiring a biopsy) method is very accurate.

From the above description of the invention, those skilled in the artwill perceive improvements, changes and modifications. Suchimprovements, changes and modifications within the skill of the art areintended to be covered by the appended claims. All references,publications, and patents cited in the present application are hereinincorporated by reference in their entirety.

Having described the invention the following is claimed:
 1. A method ofdetecting alpha-methylacyl-CoA racemase (AMACR) levels in a subject, themethod comprising: obtaining a bodily sample from the subject, thebodily sample comprising a bodily fluid selected from the groupconsisting of blood, plasma, sera, and urine, combining the bodilysample with at least one reaction solution for generating H₂O₂ uponcombination with AMACR in the bodily sample, the reaction solutionincluding a (2R)-2-methylacyl-CoA epimer that can be chirally invertedby AMACR to a (2S)-2-methylacyl-CoA epimer and an enzyme that carriesout beta oxidation with the (2S)-2-methylacyl-CoA epimer to generatehydrogen peroxide (H₂O₂); and detecting the amount of H₂O₂ generated inthe reaction solution with a biosensor, wherein increased amount of H₂O₂detected compared to a control is indicative of an increased amount ofAMACR in the bodily sample.
 2. The method of claim 1, the reactionsolution including coenzyme A (CoA), peroxisomalacyl-coenzyme A oxidase3 (ACOX3), adensonsine triphosphate (ATP), and a branched fatty acidwith (R) and (S) epimers of which only the (R) epimer is a reactionsubstrate for AMACR.
 3. The method of claim 2 the branched fatty acidcomprising pristanic acid.
 4. The method of claim 1, the biosensorincluding a working electrode and a counter electrode, the workingelectrode and counter electrode including catalyst particles forincreasing the rate of electrochemical oxidation-reduction reaction withH₂O₂ and providing the detection of H₂O₂ at a lower oxidation potentialthan without the presence of the catalyst particles.
 5. The method ofclaim 4, the catalyst particles comprising nano-particle metalliccatalysts.
 6. The method of claim 4, the catalyst particles comprising aunary metal (M), a binary metal (M-X), a unary metal oxide (MOy), abinary metal oxide (MOy-XOy), a metal-metal oxide composite material(M-MOy) or a combination of which, wherein y is less than 3, and M and Xare independently selected from a group consisting of Li, Na, Mg, Al, K,Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Sr, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag,Cd, In, Sn, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Lu, Ta,W, Os, Ir, Pt, Au, and Pb.
 7. The method of claim 4, the catalystparticles comprising iridium oxide particles.
 8. The method of claim 4,further comprising applying voltage potentials to the working electrodeand counter electrode and measuring the current flow between the workingelectrode and counter electrode to determine the level of H₂O₂.
 9. Amethod of detecting prostate cancer or an increased risk of prostatecancer in a subject, the method comprising: obtaining a bodily samplefrom the subject, the bodily sample comprising a bodily fluid selectedfrom the group consisting of blood, plasma, sera, and urine, combiningthe bodily sample with at least one reaction solution for generatingH₂O₂ upon combination with alpha-methylacyl-CoA racemase (AMACR) in thebodily sample, the reaction solution including a (2R)-2-methylacyl-CoAepimer that can be chirally inverted by AMACR to a (2S)-2-methylacyl-CoAepimer and an enzyme that carries out beta oxidation with the(2S)-2-methylacyl-CoA epimer to generate hydrogen peroxide (H₂O₂); anddetecting the amount of H₂O₂ generated in the reaction solution with abiosensor, wherein increased amount of H₂O₂ detected compared to acontrol is indicative of the subject having prostate cancer or anincreased risk of prostate cancer.
 10. The method of claim 9, thereaction solution including coenzyme A (CoA), peroxisomalacyl-coenzyme Aoxidase 3 (ACOX3), adensonsine triphosphate (ATP), and a branched fattyacid with (R) and (S) epimers of which only the (R) epimer is a reactionsubstrate for AMACR.
 11. The method of claim 10, the branched fatty acidcomprising pristanic acid.
 12. The method of claim 9, the biosensorincluding a working electrode and a counter electrode, the workingelectrode and counter electrode including catalyst particles forincreasing the rate of electrochemical oxidation-reduction reaction withH₂O₂ and providing the detection of H₂O₂ at a lower oxidation potentialthan without the presence of the catalyst particles.
 13. The method ofclaim 12, the catalyst particles comprising nano-particle metalliccatalysts.
 14. The method of claim 12, the catalyst particles comprisinga unary metal (M), a binary metal (M-X), a unary metal oxide (MOy), abinary metal oxide (MOy-XOy), a metal-metal oxide composite material(M-MOy) or a combination of which, wherein y is less than 3, and M and Xare independently selected from a group consisting of Li, Na, Mg, Al, K,Ca, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Sr, Y, Zr, Nb, Mo, Ru, Rh, Pd, Ag,Cd, In, Sn, Ba, La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Lu, Ta,W, Os, Ir, Pt, Au, and Pb.
 15. The method of claim 12, the catalystparticles comprising iridium oxide particles.
 16. The method of claim12, further comprising applying voltage potentials to the workingelectrode and counter electrode and measuring the current flow betweenthe working electrode and counter electrode to determine the level ofH₂O₂.